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  • br around the CD stained neo vasculatures Compared


    around the CD31-stained neo-vasculatures. Compared with control, the deficiency in catecholamines caused a significant reduction in MVD and M2 density in lung cancer models. After 6OHDA treatment, the MVD dropped to less than half of those in control groups in both the H446 and HCC827 models with a greater decline of M2 density in H446 (61.8%) mice than in HCC827 (46.9%) mice (Fig. 4B). These analyses collectively reveal that adrenergic signals mediated by macrophages could shift their polarization and modulate tumor angiogenesis.
    3.5. Catecholamine-stimulated macrophages contribute to angiogenesis
    The findings that catecholamine-treated macrophages promote tube formation led us to believe an underlying link exists between ca-techolamines and macrophage-associated angiogenesis in lung cancer. To explore the underlying mechanisms, the key factor, VEGF, was
    Y. Xia, et al. Brain, Behavior, and Immunity xxx (xxxx) xxx–xxx
    Fig. 5. The inhibition of adrenergic signaling restrains the catecholamine-induced VEGF secretion. (A) Release of VEGF was measured in the supernatant of EPI- and NE-treated M1 macrophages. n = 3. (B) Expression of VEGF was measured in tumor tissues after injection with 6OHDA or not. n = 3. (C) Expression of VEGF was measured in catecholamine-stimulated M1 KPT 330 and cells pretreated with propranolol. n = 3. (D) The expression of CD86 and CD206 were assessed in EPI- or NE-treated M1 macrophages preincubated with propranolol or not. n = 3. (E) The formation of capillary-like tubular structures in the presence of propranolol or not was observed in catecholamine-treated M1 macrophages. n = 3. *p < 0.05. **p < 0.01. ***p < 0.001. Abbreviations: N.S.: non-statistical significance; A.U.: arbitrary units.
    evaluated in cells and in tissues. In vitro, the VEGF secretion of mac-rophages increased significantly after incubation with catecholamines (Fig. 5A). In vivo, the concentration of VEGF reduced remarkably due to the diminished levels of catecholamines (Fig. 5B).
    To assess the relationship between VEGF and the chief adrenergic signaling of macrophages, adrenergic signaling was suppressed with the use of nonselective β-blocker propranolol to pretreat M1 macrophages before catecholamine stimulation. As expected, co-incubation with propranolol caused a significant inhibition of the catecholamine-in-duced increase of VEGF secretion in macrophages (Fig. 5C) and partly neutralized the effect of catecholamine on M2-like polarization (Fig. 5D). Inhibiting adrenergic signaling decreased the proportion of M2 cells in polarized macrophages from 90.8% to 13.2% in EPI-treated 
    cells, 93.3% to 7.4% in NE-treated cells. Results of tube formation assay also reinforced the fact that the inhibition of adrenergic signaling weakened the building of capillary-like structures (Fig. 5E), leading to reduced junctions, branches, length, and lumens. Taken together, these findings suggest that catecholamine-induced macrophages polarize to-ward M2-like phenotype to promote angiogenesis by secreting VEGF, which is regulated through adrenergic signaling.
    3.6. Catecholamines alter innate immune cell distribution within the tumor microenvironment
    Macrophages regulate the turnover of immune response not only by the release of pro-or anti-tumor factors, but also by the communication
    Y. Xia, et al. Brain, Behavior, and Immunity xxx (xxxx) xxx–xxx
    percentage of intratumoral active DCs from 12.2% to 34.6% in the former and kept roughly equal in the latter (Fig. 6C, P = 0.038 for H446, P = 0.55 for HCC827). Despite no significance in the rare NKs (P = 0.30 for H446, P = 0.09 for HCC827), which only accounted for 1–2% or less in the tumor microenvironment, the average proportion was still higher in H446 mice, the more effective model after 6OHDA treatment (Fig. 6B). These data collectively imply that inhibition of adrenergic signaling impacts on multiple immune cells, remodeling a more defensive tumor microenvironment via the neuro-immune axis.
    4. Discussion
    In this study, we reported that catecholamines could reshape the TAM phenotype and reeducate them toward tumor-supportive M2-po-larized macrophages by stimulating adrenergic signaling. These find-ings emphasize the plasticity of the TAM phenotype induced by ca-techolamines and the possibility to revert their antitumor properties through dampening of adrenergic nerve activity. Consequently, adre-nergic receptors on macrophages have emerged as an interest in cancer
    biology due to the findings linking neurotransmitters and macrophage functions. Consistent with previous reports about the pro-tumoral ef-fects of M2-polarized macrophages (Hughes et al., 2015; Sica et al., 2006), our data also revealed that the stimulation of ARs on macro-phages resulted in the production of pro-angiogenic molecules and immunosuppressive cytokines, while suppressing proinflammatory cy-tokines, leading to a switch of immune dysfunction. Im-munosuppressive IL-10 were potently upregulated after catecholamine treatment with a decreased release of proinflammatory IL-6 and IL-12, facilitating the immune escape of tumor cells.